Ischemic brain injury due to cardiac arrest or stroke represents a major cause of mortality and disability. Although the precise mechanisms of delayed post-ischemic neurodegeneration remain incompletely understood, disruption of Ca2+ homeostasis appears to play a major role. One potential cause of disrupted calcium homeostasis in post-ischemic neurons is proteolytic modification of Ca2+ regulatory proteins. This proposal focuses on caspase 3- and calpain-mediated cleavage of the inositol (1,4,5)-trisphosphate receptor (IP3R), a Ca2+ release channel located on the endoplasmic reticulum (ER). Both published evidence and our own preliminary data suggest that caspase 3- and calpain-mediated cleavage of the type 1 IP3R (IP3R1) generates a constitutively open channel that allows Ca2+ to leak from the ER and impairs the ER capacity to buffer cytosolic calcium overload. The aims of this proposal will test the hypothesis that caspase 3- or calpain- mediated cleavage of IP3R1 generates a constitutively open channel that irreversibly disrupts intracellular Ca2+ homeostasis and contributes to neurodegeneration after excitotoxic and ischemic injury. Specific Aim 1 will measure the channel properties of caspase 3- and calpain-cleaved of IP3R1 and their effect on intracellular calcium homeostasis. Specific Aim 2 will investigate the effect of caspase 3- and calpain-cleaved IP3R1 in primary neuron culture under baseline conditions and after excitotoxic injury. Specific Aim 3 will investigate the effect of caspase 3- and calpain-cleaved IP3R1 on neurons in vivo under baseline and post-ischemic conditions. Overall, the results of these experiments with provide critical insight into the mechanism by which pathologic proteases cause acute neurodegeneration through disruption of intracellular calcium homeostasis. In addition, blocking the caspase- and calpain-cleaved forms of IP3R1 could be a novel therapeutic target for neuroprotection after ischemic brain injury. PUBLIC HEALTH RELEVANCE: A growing body of evidence suggests that sustained disruption of neuronal calcium homeostasis plays a causal role in neuronal death after brain ischemia. This proposal tests the hypothesis that pathologic proteases, caspase-3 and calpains, disrupt neuronal calcium homeostasis through cleavage of the inositol (1,4,5)-trisphosphate receptor, a Ca2+ channel located on the endoplasmic reticulum. The results of these experiments with provide fundamental insight into the mechanism of post-ischemic neurodegeneration and potentially identify a novel therapeutic target for neuroprotection after ischemic brain injury.